Abstract
Sec1/Munc18-family (SM) proteins are required for SNARE-mediated membrane fusion, but their mechanism(s) of action remain controversial. Using single-molecule force spectroscopy, we found that the SM protein Munc18-1 catalyzes step-wise zippering of three synaptic SNAREs (syntaxin, VAMP2, and SNAP-25) into a four-helix bundle. Catalysis requires formation of an intermediate template complex in which Munc18-1 juxtaposes the N-terminal regions of the SNARE motifs of syntaxin and VAMP2, while keeping their C-terminal regions separated. SNAP-25 binds the templated SNAREs to induce full SNARE zippering. Munc18-1 mutations modulate the stability of the template complex in a manner consistent with their effects on membrane fusion, indicating that chaperoned SNARE assembly is essential for exocytosis. Two other SM proteins, Munc18-3 and Vps33, similarly chaperone SNARE assembly via a template complex, suggesting that SM protein mechanism is conserved.
Highlights
Cytosolic Sec1/Munc18 (SM) proteins and membrane-anchored SNARE proteins constitute the core machinery that mediates most intracellular membrane fusion (Rizo and Sudhof, 2012; Sudhof and Rothman, 2009)
We found that the SM protein Vps33 forms binary complexes with the SNARE motifs of Vam3 (Qa-SNARE) and Nyv1 (R-SNARE), as well as a ternary complex containing all three proteins (Baker et al, 2015)
Crystal structures of the two binary complexes revealed that the Qa-SNARE and the R-SNARE bind to adjacent sites on the SM protein and led to a model of the template complex in which the two SNARE motifs are ‘half-zippered’
Summary
Cytosolic Sec1/Munc (SM) proteins and membrane-anchored SNARE proteins constitute the core machinery that mediates most intracellular membrane fusion (Rizo and Sudhof, 2012; Sudhof and Rothman, 2009). Related SM proteins, Munc and Munc, are required for cytotoxin release from lymphocytes to kill cancerous or infected cells (Cote et al, 2009) and for glucose uptake (Bryant and Gould, 2011), respectively. SM proteins regulate the assembly of SNAREs into the membrane-bridging ‘trans-SNARE’ complexes required for membrane fusion (Figure 1) (Baker and Hughson, 2016; Brunger et al, 2018; Gao et al, 2012; Rizo and Sudhof, 2012; Shen et al, 2007; Sudhof and Rothman, 2009; Sutton et al, 1998).
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